Passion fruit seed extract protects beta‐amyloid‐induced neuronal cell death in a differentiated human neuroblastoma SH‐SY5Y cell model

Abstract Alzheimer's disease (AD) is a progressive neurodegenerative disease with accompanying perceptive disorder. We previously reported that decreasing levels of brain‐derived neurotrophic factor (BDNF) promoted beta‐amyloid (Aβ)‐induced neuronal cell death in neuron‐like differentiated SH‐SY5Y (ndSH‐SY5Y) human neuroblastoma cells in an AD mimic cell model. We investigated the neuroprotective effects of passion fruit seed extract (PFSE) and one of the main stilbene compounds, piceatannol, in an AD cell model using ndSH‐SY5Y cells. Both PFSE and piceatannol were found to protect Aβ‐induced neurite fragmentation in the cell model (protection efficacy; 34% in PFSE and 36% in piceatannol). In addition, both PFSE and piceatannol suppress Aβ‐induced neuronal cell death in the cell model (inhibitory effect; 27% in PFSE and 32% in piceatannol). Our study is the first to report that piceatannol‐rich PFSE can repress Aβ‐induced neuronal cell death by protecting against neurite fragmentation in the AD human cell model. These findings suggest that piceatannol‐rich PFSE can be considered a potentially neuroprotective functional food for both prevention and treatment of AD.


| INTRODUC TI ON
Alzheimer's disease (AD) is a progressive neurodegenerative disease with accompanying perceptive dysfunction, irreversible memory loss, disorientation, and language impairment (Lane et al., 2018;Liu-Seifert et al., 2015;Scheff et al., 2006). Beta-amyloid (Aβ) accumulation is widely considered the main neurotoxic injury that causes AD (Duyckaerts et al., 2009;Fricker et al., 2018;Lane et al., 2018;Mucke & Selkoe, 2012;Scheltens et al., 2016). The common neuropathological features of AD include neurofibrillary tangles comprising hyperphosphorylated tau and senile plaques deposited on Aβ peptides (Duyckaerts et al., 2009;Fricker et al., 2018;Lane et al., 2018;Scheltens et al., 2016). In patients with AD, both brain and serum levels of brain-derived neurotrophic factor (BDNF)-a neurotrophin widely distributed in adult brainshave been reported to be significantly decreased compared with healthy individuals (Connor et al., 1997;Ng et al., 2019). BDNF plays an important role in the pathophysiology of Aβ-induced neurotoxicity in AD (Acheson et al., 1995;Arancio & Chao, 2007). We previously demonstrated that the decrease in BDNF triggers selfaggregated Aβ-induced neurite fragmentation as well as two types of neuronal cell death, namely caspase-6-dependent apoptosis and necroptosis, in an AD mimic cell model using neuron-like differentiated SH-SY5Y (ndSH-SY5Y) human neuroblastoma cells (Tagai et al., 2020). In addition, we have been studying the neuroprotective effects of several natural products and compounds by using an AD mimic cell model.
Passion fruit (Passiflora edulis Sims) is a tropical plant belonging to the Passifloraceae family, whose fruits are commonly consumed across the world because of its attractive aroma, flavor, and taste (He et al., 2020). Passion fruit seed extract (PFSE) has a potential to become a functional food because of being a rich source of stilbenes, especially piceatannol (3,3′,4′,5-trans-tetrahydroxy-stilben e) (Matsui et al., 2010). Piceatannol is a naturally occurring stilbene derivative and a structurally related polyphenol analog of resveratrol (3,4′,5-trans-trihydroxy-stilbene). Several previous studies showed that piceatannol possessed neuroprotective effects. Monti et al. (Rivière et al., 2007) indicated that stilbenes, including resveratrol and piceatannol, inhibited Aβ peptide aggregation in vitro. Kim et al. (2007) reported that piceatannol prevented Aβ-induced reactive oxygen species (ROS) accumulation and apoptosis in PC12 rat pheochromocytoma cells. In addition, Lu et al. (2016) indicated that two stilbenoids, namely piceatannol and pterostilbene, activated the PI3K/Akt/Bad signaling pathway and suppressed both Aβ-induced ROS accumulation and apoptotic cell death in PC12 cells. Furthermore, a previous study has shown that stilbenoids, including resveratrol and piceatannol, suppress both Aβ-induced neurotoxicity and cell death by decreasing intracellular ROS via the PI3K/Akt signaling pathway in rat primary cortex neurons (Wen et al., 2018). Although piceatannol has been reported to possess neuroprotective effects, these effects have not been studied before on PFSE. In this study, we have focused on the bioactivities of piceatannol and piceatannol-rich PFSE as a functional food in both healthcare and preventive care. We previously reported that piceatannol and/or PFSE showed a variety of biological activities such as skin protection (Maruki-Uchida et al., 2013;Matsui et al., 2010), vasodilatation (Kinoshita et al., 2013), chronic diseases' prevention (Kawakami et al., 2014), and metabolic improvement (Kitada et al., 2017;Uchida-Maruki et al., 2015;Yamamoto et al., 2017). Interestingly, in our previous study, we indicated that PFSE has suppressed cancer cell proliferation more intensely than piceatannol in both NCI-H522 human non-small-cell lung cancer cells and HCT116 human colorectal cancer cells (Yamamoto et al., 2019).
In this study, we analyzed the neuroprotective effects of piceatannol-rich PFSE and piceatannol in an AD cell model using human neuroblastoma neuron-like differentiated cell model . In addition, we discussed the potentially neuroprotective functional foods prepared using PFSE in AD prevention and treatment.

| Reagents
Piceatannol was purchased from Tokyo Chemical Industry.
Piceatannol was stored in 100 mM stocks in dimethyl sulfoxide

| PFSE preparation
Piceatannol-rich PFSE was prepared as previously described (Yamamoto et al., 2019). Briefly, passion fruit seeds were milled, lyophilized, and extracted with 35% ethanol. After centrifuging the extract, the supernatant was evaporated and lyophilized to obtain a PFSE powder. PFSE, dissolved in DMSO, was stored in 100 mg/ml stocks at −20°C.

| Cell culture and cell differentiation
The SH-SY5Y human neuroblastoma cell line (ATCC ® CRL-2266 TM ) was purchased from the American Type Culture Collection. Cell culture and differentiation were performed as described previously (Tagai et al., 2020). In brief, SH-SY5Y cells were cultured in Dulbecco's modified eagle medium (D-MEM)/Ham's F-12 (FUJIFILM Wako Pure Chemical Corporation, Cat#048-29785) containing 10% fetal bovine serum (FBS), 100-units/ml penicillin, and 100μg/ ml streptomycin in a 37°C incubator in an atmosphere of 5% CO 2 and 100% relative humidity. In the cell differentiation, SH-SY5Y cells were inoculated at an initial density of 1.0 × 10 5 cells/dish in a collagen I-coated φ 3.5 cm dishes. All-trans-RA (FUJIFILM Wako) was added 2 days after plating at a final concentration of 10 μM dis-

| Cell viability
Neuron-like differentiated SH-SY5Y (i.e., ndSH-SY5Y) cells in several developmental stages were stained with 7-aminoactinomycin D (7-AAD; Thermo Fisher Life Technologies) and Hoechst33342 (Thermo Fisher Life Technologies). Cell images were analyzed using a LAS AF microscope coupled with a Leica DMI6000B-AFC system at ×200 magnification, and calculations were performed based on the proportion of 7-AAD-positive cells.

| Morphological observation
Cell morphology was observed under a Leica DMi1 microscope using a LAS v4.12 platform at ×200 magnification. Measurements of both neurite length and neurite fragmentation were analyzed using the image processing software ImageJ using the NeuronJ plugin and Particle Analysis. Neurite fragments were measured as follows: image binarization followed by automatic calculation by particle analysis using appropriate parameters (size: 0.00005-0.0005; circularity: 0.40-1.00).

| Statistical analysis
Statistical analysis was performed using GraphPad Prism 9 software.
Data were presented as means ±standard deviation (SD). The significance of the differences among groups was determined using the Student's t-test and one-way ANOVA. p < .05 was considered as significant.

| PFSE and piceatannol prevent Aβ-induced neurite fragmentation
We previously reported an AD mimic cell model using ndSH-SY5Y cells (Table 1) (Tagai et al., 2020). We also showed that both Aβinduced neurite fragmentation and cell death modes were modified under reducing concentrations of BDNF (Tagai et al., 2020). In this study, to examine the neuroprotective effects of piceatannol-rich PFSE and piceatannol, we analyzed their effects on Aβ-induced neurite fragmentation and cell death in an AD cell model using ndSH-SY5Y cells. PFSE was found to contain piceatannol, as the most abundant polyphenol, at a concentration of 104.5 μg/mg (Table 2) (Yamamoto et al., 2019). Figure 1 indicates the chemical structures of the main polyphenol content in PFSE. In addition, Figure 2 shows the scheme of experimental procedure in the ndSH-SY-5Y cell model. This evaluation method differentiates cells and is not suitable for the CCK-8 assay on a 96-well plate. Cells were treated for 5 days (days 2-7) with 10-μM RA before incubation with 100 ng/ml BDNF for 2 days (days 7-9). On the basis of the morphological observations, ndSH-SY5Y cells ceased to proliferate on day 5, and their morphology became neuron like, including the development of long neurites that were visible on days 5-9 (Tagai et al., 2020). It should be noted that the neurite length of ndSH-SY5Y cells was approximately 87 μm on day 9 (Tagai et al., 2020). On day 9, ndSH-SY5Y cells expressed growth-associated protein 43 (GAP43), a neuron-specific marker protein (Table 1) (Tagai et al., 2020).
At first, we examined the protective effects of PFSE and piceatannol on neurite fragmentation in an AD cell model. Neurite fragmentation was only scarcely observed when BDNF levels were Note: Polyphenols present in PFSE were analyzed as described previously (Yamamoto et al., 2019). Briefly, PFSE was analyzed via multiple-reaction monitoring combined with ultrafast liquid chromatograph and electrospray ionization tandem mass spectrometry system. decreased ( Figure 3). Thus, treatments of ndSH-SY5Y cells with either 10 or 100 ng/ml BDNF for 96 h on day 9 did not change cells' morphology at day 13 ( Figure 3a). As shown in Figure

| PFSE and piceatannol suppress Aβ-induced neuronal cell death
In a previous study, we demonstrated that neuronal cell death, induced by both aggregated Aβ and the low levels of BDNF, is characterized as caspase-6-dependent apoptosis and necroptosis in an AD cell model (Table 1)  approximately 60% or 56% after the treatment with PFSE or piceatannol, respectively (Figure 4). We also showed that PFSE and piceatannol depress Aβ-induced ndSH-SY5Y cell death at similar levels.
These results indicated that both piceatannol-rich PFSE and piceatannol were able to suppress Aβ-induced neuronal cell death in an AD cell model.

| DISCUSS ION
Numerous studies have been performed on functional foods for Alzheimer's disease, namely foods or nutrients that provide medical or health benefits, including prevention and treatment of the disease (Atlante et al., 2020). Several functional foods and nutraceutical compounds act at a variety of biochemical and metabolic levels, and much evidence shows their neuroprotective effects in fundamental studies, that is, in vitro and in vivo experiments (Atlante et al., 2020).
In this study, we focused on the biological effects of piceatannolrich PFSE and piceatannol in order to be evaluated as a functional food in healthcare, preventive care, and diverse chronic disease treatment. It was previously suggested that PFSE powder has the potential to be used as a functional food, being a rich source of stilbenes, especially piceatannol (Matsui et al., 2010). In the present study, we showed that both piceatannol and piceatannol-rich PFSE were able to prevent Aβ-induced neurite fragmentation and neuronal cell death in an AD cell model using neuron-like differentiated human SH-SY5Y Several previous studies demonstrated that piceatannol exhibited neuroprotective effects on Aβ-induced neurotoxicity in both rat pheochromocytoma PC12 cells and rat primary cortex neurons (Fu et al., 2016;Kim et al., 2007;Wen et al., 2018). In the amyloid cascade hypothesis of AD onset, deposition of neurotoxic Aβ in the brain is regarded as an important reason of AD pathogenesis (Lu et al., 2013).
Aβ peptides are processed by the proteolysis of the amyloid precursor protein by β-and γ-secretase (Benilova et al., 2012). Many previous studies have confirmed that Aβ neurotoxicity is associated with oxidative stress (Butterfield et al., 2007;Cai et al., 2011;Cheignon et al., 2018). As reported previously, piceatannol inhibited both Aβinduced ROS accumulation and caspase-3-dependent apoptosis in PC12 cells (Kim et al., 2007). In addition, Li et al. (Fu et al., 2016) demonstrated that piceatannol repressed both Aβ-induced apoptosis, mediating the PI3K/Akt/Bad signaling and downstream mitochondria-caspase-3-dependent cell death pathway in PC12 cells. Furthermore, piceatannol suppressed Aβ-induced neurotoxicity by decreasing intracellular ROS accumulation via the PI3K/Akt signaling pathway in rat primary cortex neurons (Wen et al., 2018) and prevented Aβ-induced intracellular ROS accumulation in both rat PC12 cells and rat primary cortex neurons (Fu et al., 2016;Wen et al., 2018). Interestingly, our collaborators previously revealed that piceatannol promoted neural stem cell differentiation into astrocytes (Arai et al., 2016). Notably, oral administration of piceatannol increased the proportion of astrocytes in the brains of adult mice (Arai et al., 2016). These findings suggest that piceatannol can successfully pass through the blood-brain barrier. Importantly, we further investigated the neuronal cell death mode(s) and the detail action mechanisms of PFSE in an AD mimic cell model. In this regard, we are currently investigating cell death and amyloidosis marker proteins in an AD cell model via comprehensive proteomic analysis. We also realize that additional primary human neuronal cell-based and in vivo animal studies are warranted to verify the efficacies of PFSE and piceatannol.
In conclusion, piceatannol-rich PFSE can be considered a promising neuroprotective functional food in the maintenance of cerebral function as well as in the prevention and treatment of AD.

ACK N OWLED G EM ENTS
We thank Ms. Ayako Tanaka (Tokyo University of Science) for the helpful discussions.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.